This invention relates to screening methods for cells and for molecules of interest. More particularly, this invention relates to methods of isolating a mutant cell which expresses a desired compound utilizing lambdoid bacteriophage infection and the formation of a symbiotic relationship between two different cell types.
It is common to isolate mutant cells which overproduce a specific metabolite by selecting cells which are resistant to analogs of the metabolite. For example, Yamada et al. (Agric. Biol. Chem. (1983) 47:1011) describe the isolation of mutants which overproduce biotin by selecting for cells resistant to a biotin analog. Yamada et al. (Agric. Biol. Chem. (1982) 46:47) and Chattopadhyay et al. (J. Gen. Microbiol (1991) 137:685) isolated methionine overproducing mutants by selecting cells resistant to ethionine. Hifferd (Iowa State J. Res. (1988) 62: 479) isolated valine-overproducing mutants, selecting for valine resistance. Kempe et al. (Cell (1976) 9:541) isolated pyrimidine nucleotide biosynthetic enzyme overproducers, selecting for resistance to N-phosphonoacetyl-l-aspartate. Hall et al. (J. Bactriol. (1989) 143:981), isolated amino acid overproducers in cyanobacteria by selecting for antimetabolite analog resistance. In Hall et al. (ibid. ) wild type Bacillus subtilis cells were used as a test lawn for screening obvious regulatory mutants from among collections of analog resistant strains. Auxotrophic strains of B. subtillis were convenient indicator strains for identification of mutants in Cyanobacteria through observation of syntrophic growth responses. Green (2d Chem. Congress N. Am. Continent, San Francisco, Aug. 22, 1989, Abst. No. 16) describes production of mutant corn cells resistant to lysine- and threonine-induced growth inhibition. Grull et al. (J. Bacteriol. 1 (1979) 137:480) describe isolation of amino acid overproducing mutants of Escherichia coli (E. coli) obtained by mutagenesis and penicillin enrichment. Vincenzotto et al. (Arch. Internat. de Chemi (1982) 90:B88) describe isolation of mutants of an alga by mutagenesis and screening on agar medium containing various dyes. Santhaguru et al.(Israel J. Med. Sci. (1985) 21:185) describe use of levulinate, a competitive inhibitor of the heme biosynthetic pathway, for isolation of heme overproducing Rhizobium mutants.
Isolation of cells that produce a desired compound from a population of cells that do not make the desired compound is a problem when the production of the desired compound does not endow the cell with any selective advantage. In these instances, a method of screening rather than selecting for production of the desired product must be developed. A screening method which employs a detector strain present in an overlay was utilized by Pai (J. Bacteriol. (1972) 112:1280) to isolate a strain that overproduces biotin. In this method, wild type E. coli were mutagenized and plated with an E. coli biotin auxotroph. The mutagenized E. coli was not itself an auxotroph, and thus, a mutual two-way symbiotic relationship was not achieved. A similar method has more recently been employed to screen for lysine excretors (LiMuti et al. (1989) Microbiol Meth. 9:129). A variation of the overlay method has been developed where a micropore membrane is used to separate the strain from the substrate (U.S. Pat. No. 4,421,849). A problem with these strategies is the limitation on the number of cells that can be screened at one time. The limit is based on the need to distinguish individual colonies on a plate which places the limit, e.g. for bacteria, at 1000 to 10,000 per each 100 mm diameter plate. If the event resulting in production of the desired compound occurs at a frequency of 10 .sup.-7, then 1000 plates would have to be screened to obtain one event.
Bacteriophages have been used in strategies for detecting desired compounds. For example, a method employing the bacteriophage M13 has been used to assay for various proteins of interest. In this method, M13 bacteriophage displaying peptides fused to pIII, a minor M13 coat protein, have been used to screen for protein binding molecules and antibodies (Scott et al. (1990) Science 249:386; Devlin et al. (1990) Science 249:404). Special M13-derived systems have been used to express antibodies as fusion proteins on the surface of the bacteriophage, and techniques have been developed to enrich the population for bacteriophage expressing antibodies with desired affinities for an antigen (Garrard et al. (1991) Bio/Technol. 9:1373; Barbas et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:7978). However, the use of M13 in assay methods is limited because M13 infection is not immediately ascertainable. This is because infection by M13 does not provide the cell with compounds required for growth and is not lytic.
Like M13, T4 has been used in assays for various proteins such as nerve growth factor (NGF) (Oger et al. (1974) Proc. Natl. Acad. Sci. (USA) 71:1554-1558). In this assay, T4 was chemically coupled to NGF using glutaraldehyde. The bacteriophage was then rendered non-infective by treatment with antibodies against NGF. When unbound NGF was added to the medium, NGF-linked bacteriophage was displaced from the antibody and became free to infect E. coli. Bacteriophage T4 has also been used to detect antibodies against a wide range of compounds. For example, Becker et al. (Immunochem. (1970) 7:741) used a T4 bacteriophage to detect antibodies against p-azobenzenearsonate. Hurwitz et al. (Eur. J. Biochem. (1970) 17:273) used a T4 bacteriophage to detect and estimate levels of angiotensin-II-beta-amide and its antibodies. Gurari et al. (Eur. J. Biochem. (1972) 26:247) used bacteriophage T4 in the detection of antibodies to nucleic acids. These detection methods involve the chemical modification of the T4 bacteriophage resulting in the non-specific exposure on the bacteriophage surface of a compound to which the antibodies to be assayed are targeted. Such antibodies render the bacteriophage non-infective, thus enabling the decrease in plaque formation to be used as a measure of the level of antibody present. The T4 system has also been used to measure hapten concentrations (see, e.g., Hurwitz et al. (1970) Eur. J. Biochem. 17:273-277) In this system, T4 is chemically modified such that it exposes the desired hapten non-specifically on its surface. The addition of anti-hapten antibody blocks the infectivity of the bacteriophage. Infectivity is restored in the presence of hapten.
Although both the M13 and T4 bacteriophage systems can be used to detect the presence of a desired compound (or a cell producing that compound) by their ability to become infectious in the presence of that compound (and thus in the presence of a cell producing that compound), infection by M13 is normally not immediately ascertainable, and T4 infection is lethal. Thus, these systems cannot be used where a quick selection method based on the survival of the infected bacterial cell is desired, such as where a particular cell type is being selected, or when the object of bacteriophage infection is to restore the ability of an auxotrophic bacterial cell to survive on its own under a given set of growth conditions. Special M13-derived phagemid systems carry genes which could endow an infected cell with a selective growth advantage (Barbas et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:7978). However, these systems have not been used to detect a desired compound or cells producing such compounds. Furthermore, because gpIII, the M13 protein to which the target molecules are fused, accumulates on the inner membrane facing the periplasm, there are limitations on the nature of the protein fusion. Fusions that are not able to cross the membrane will not be assembled into M13. In addition, in all M13 systems where fusion proteins have been used to display proteins on the outer surface, the displayed protein (or peptide) itself has been the desired compound.
Thus, what is needed are new methods for selecting cells that excrete desired compounds. What is also needed are selection methods that do not limit the desired compounds to those on which cell growth is dependent. In addition, what is needed are selection methods that do not limit the desired compounds to those that can be assayed based on changes in color, turbidity or viscosity. Finally, what are needed are selection methods that are adaptable to a broad range of cell types.